DNA sequence analysis techniques have evolved to efficiently handle large scale sequencing projects. However, there are limitations in the currently available techniques when applied to high throughput sequencing projects where it is desirable to limit costs and retain sufficient speed. For example, classic Sanger dideoxy sequencing methods employ a step of resolving DNA fragments on a gel. This step does not lend itself to very large-scale multiplexing or parallel processing and further, has the problem of band compression during electrophoresis. Other techniques have been developed to increase the speed and decrease the cost of sequencing. These include sequencing by hybridization (see, e.g., Bains and Smith, J. Theoret. Biol. 135:303-307, 1988; Drmanac et al. Genomics 4:114-128, 1989; Khrapko et al. FEBS Lett. 256:118-122, 1989; Southern, WO10977, 1989); parallel signature sequencing based on ligation and cleavage (e.g., Brenner et al. Proc. Natl. Acad. Sci. 97:1665-1670, 2000); sequencing using reversible chain terminating nucleotides (see, e.g., U.S. Pat. Nos. 5,902,723 and 5,547,83; Canard and Arzumanov, Gene 11:1 (1994); and Dyatkina Arzumanov, Nucleic Acids Symp Ser 18:117 (1987)); reversible chain termination with DNA ligase (see, e.g., U.S. Pat. No. 5,403,708); time resolved sequencing (see, e.g., Johnson et al., Anal. Biochem. 136:192 (1984); and pyrosequencing (e.g., Ronaghi et al. Anal. Biochem 242:84-89, 1996).
Pyrosequencing is based on the concept of sequencing by synthesis (e.g., U.S. Pat. No. 4,863,849). The technique can be applied to massively parallel sequencing projects. For example, using an automated platform, it is possible to carry out hundreds of thousands of sequencing reactions simultaneously. Sequencing by synthesis differs from the classic dideoxy sequencing approach in that instead of generating a large number of sequences simultaneously and then characterizing them at a later step, real time monitoring of the incorporation of each base into a growing chain is employed. Although this approach is slow in the context of an individual sequencing reaction, it can be used for generating large amounts of sequence information in each cycle when hundreds of thousands to millions of reactions are performed in parallel. Despite these advantages, there are still limitations in the pyrosequencing approach. For example, there are difficulties in determining the number of incorporated nucleotides in homopolymeric regions, due to the nonlinear signal response following the incorporation of multiple identical molecules. Other Sequencing by Synthesis approaches on solid phase arrays that do not employ reversible terminators have similar disadvantages.
A method of sequencing using chemically reversible terminators using 3′-O-Allyl modified nucleotide analogs has recently been described (Ruparel et al., Proc. Natl. Acad. Sci. 102:5932-5937, 2005). In this method, the nucleotide analog contains an allyl moiety that caps the 3′-OH group and a fluorophore linked to the 5′ position of the uracil through a photocleavable linker. This nucleotide is a substrate for a DNA polymerase. After incorporation into a DNA strand and photocleavage of the linker, the allyl group is removed using a Pd-catalyzed reaction, and the polymerase reaction is reinitiated. Thus, these analogs can act as reversible terminators in sequencing by synthesis reactions. Other reversible terminators are described, e.g., in U.S. Pat. Nos. 5,872,244; 6,232,465; 6,214,987; 5,808,045; 5,763,594, and 5,302,509; and U.S. Patent Application Publication No. 20030215862. The blocked 3′-OH type of reversible terminators suffer from several drawbacks including poor incorporation and deblocking efficiencies and the tedious conditions used for deblocking. A highly desirable method for high throughput sequencing based on reversible terminators demonstrates near perfect incorporation, chain termination, and deblocking efficiencies in order to minimize problems and background signals from out of phase reactions.
Recently, 2′-modified (e.g., 2′-phosphate) nucleoside 5′ triphosphates have been described that can be used as substrates for certain nucleic acid polymerizing enzymes for only a single base incorporation (see, e.g., US Patent Application Publication Nos. 2005/00373898 and 2005/0037991). The present invention provides new methods of sequencing and genotyping that use 2′-terminator nucleotides in a reversible termination sequencing reaction.